The present invention relates to a novel cell cycle gene in plants and to a method for controlling or altering growth characteristics of a plant and/or a plant cell comprising introduction and/or expression of one or more cell cycle regulatory protein functional in a plant or parts thereof and/or one or more nucleic acid sequence encoding such proteins. Optionally, said sequences are placed under the control of a foreign control sequence in said plant and/or plant cell.
Also provided in the present invention is a method for modulating endoreduplication in plants, plant cells or parts thereof, by genetic engineering techniques. In a preferred embodiment endoreduplication in plants, plant cells or parts thereof is modulated by modifying the plant cell cycle.
Cell division is fundamental for growth in humans, animals and plants. Prior to dividing in two daughter cells, the mother cell needs to replicate its DNA. The cell cycle is traditionally divided into 4 distinct phases:
G1: the gap between mitosis and the onset of DNA synthesis;
S: the phase of DNA synthesis;
G2: the gap between S and mitosis.
M: mitosis, the process of nuclear division leading up to the actual cell division.
The distinction of these 4 phases provides a convenient way of dividing the interval between successive divisions. Although they have served a useful purpose, a recent flurry of experimental results, much of it as a consequence of cancer research, has resulted in a more intricate picture of the cell cycle""s xe2x80x9cfour seasonsxe2x80x9d (K. Nasmyth, Science 274, 1643-1645, 1996; P. Nurse, Nature, 344, 503-508, 1990)
The underlying mechanism controlling the cell cycle control system has only recently been studied in greater detail. In all eukaryotic systems, including plants, this control mechanism is based on two key families of proteins which regulate the essential process of cell division, namely protein kinases (cyclin dependent kinases or CDKs) and their activating associated subunits, called cyclins. The activity of these protein complexes is switched on and off at specific points of the cell cycle. Particular CDK-cyclin complexes activated at the G1/S transition trigger the start of DNA replication. Different CDK-cyclin complexes are activated at the G2/M transition and induce mitosis leading to cell division.
Each of the CDK-cyclin complexes execute their regulatory role via modulating different sets of multiple target proteins. Furthermore, the large variety of developmental and environmental signals affecting cell division all converge on the regulation of CDK activity. CDKs can therefore be seen as the central engine driving cell division.
In animal systems and in yeast, knowledge about cell cycle regulations is now quite advanced. The activity of CDK-cyclin complexes is regulated at five levels: (i) transcription of the CDK and cyclin genes; (ii) association of specific CDK""s with their specific cyclin partner; (iii) phosphorylation/dephosphorylation of the CDK and cyclins; (iv) interaction with other regulatory proteins such as SUC1/CKS1 homologues and cell cycle kinase inhibitors (CKI); and (v) cell cycle phase-dependent destruction of the cyclins and CKIs.
The study of cell cycle regulation in plants has lagged behind that in animals and yeast. Some basic mechanisms of cell cycle control appear to be conserved among eukaryotes, including plants. Plants were shown to also possess CDK""s, cyclins and CKI""s. However plants have unique developmental features which are reflected in specific characteristics of the cell cycle control. These include for instance the absence of cell migration, the formation of organs throughout the entire lifespan from specialized regions called meristems, the formation of a cell wall and the capacity of non-dividing cells to re-enter the cell cycle. Another specific feature is that many plant cells, in particular those involved in storage (e.g. endosperm), are polyploid due to rounds of DNA synthesis without mitosis. This so-called endoreduplication is intimately related with cell cycle control.
Due to these fundamental differences, multiple components of the cell cycle of plants are unique compared to their yeast and animal counterparts. For example, plants contain a unique class of CDKs, such as CDC2b in Arabidopsis, which are both structurally and functionally different from animal and yeast CDKs.
The further elucidation of cell cycle regulation in plants and its differences and similarities with other eukaryotic systems is a major research challenge. Strictly for the case of comparison, some key elements about yeast and animal systems are described below in more detail.
As already mentioned above, the control of cell cycle progression in eukaryotes is mainly exerted at two transition points: one in late G1, before DNA synthesis, and one at the G2/M boundary. Progression through these control points is mediated by cyclin-dependent protein kinase (CDK) complexes, which contain, in more detail, a catalytic subunit of approximately 34-kDa encoded by the CDK genes. Both Saccharomyces cerevisiae and Schizosaccharomyces pombe only utilise one CDK gene for the regulation of their cell cycle. The kinase activity of their gene products p34CDC2 and p34CDC28 in Sch. pombe and in S. cerevisiae, respectively, is dependent on regulatory proteins, called cyclins. Progression through the different cell cycle phases is achieved by the sequential association of p34CDC2/CDC28 with different cyclins. Although in higher eukaryotes this regulation mechanism is conserved, the situation is more complex since they have evolved to use multiple CDKs to regulate the different stages of the cell cycle. In mammals, seven CDKs have been described, defined as CDK1 to CDK7, each binding a specific subset of cyclins.
In animal systems, CDK activity is not only regulated by its association with cyclins but also involves both stimulatory and inhibitory phosphorylations. Kinase activity is positively regulated by phosphorylation of a Thr residue located between amino acids 160-170 (depending on the CDK protein). This phosphorylation is mediated by the CDK-activating kinase (CAK) which interestingly is a CDK/cyclin complex itself. Inhibitory phosphorylations occur at the ATP-binding site (the Tyr15 residue together with Thr14 in higher eukaryotes) and are carried out by at least two protein kinases. A specific phosphatase, CDC25, dephosphorylates these residues at the G2/M checkpoint, thus activating CDK activity and resulting in the onset of mitosis.
CDK activity is furthermore negatively regulated by a family of mainly low-molecular weight proteins, called cyclin-dependent kinase inhibitors (CKIs). Kinase activity is inhibited by the tight association of these CKIs with the CDK/cyclin complexes.
The SUC1/CKS1 proteins represent another class of components of CDK complexes. The SUC1 and CKS1 genes were originally identified in Sch. pombe and S.cerevisiae, respectively as suppressors of certain temperature-sensitive CDC2/CDC28 alleles. Mutant p34CDC2 proteins suppressible by SUC1 overexpression were shown to have a reduced affinity for the SUC1 protein. Homologues of SUC1/CKS1 have since then been identified in a wide range of organisms, including human, Drosophila and Xenopus. The conserved interaction between SUC1/CKS1 proteins with CDKs allows purification of homologous CDKs from other species using affinity chromatography.
More than one decade after their initial discovery, the function of the SUC1/CKS1 genes is still not resolved. In yeasts, both SUC1 and CKS1 are essential genes, as was demonstrated by gene disruption. Cells deleted for SUC1 show mitotic spindles of varying lengths and condensed chromosomes, typical for a late mitotic arrest. The presence of high cyclin levels suggests that this arrest is attributed to the inability to destroy the mitotic cyclins, which is a prerequisite to leave M phase. Mitotic cyclins are normally destroyed by the ubiquitin-dependent proteosomal pathway. An essential component in this destruction pathway is a multiprotein complex called the anaphase-promoting complex (APC) or cyclosome. Mutations in the APC result in a stabilisation of mitotic cyclins and cause an anaphase arrest.
However, in addition, the presence of high concentrations of SUC1/CKS1 blocks cell cycle progression. Analysis of Xenopus cell-free extracts indicates that the high SUC1/CKS1 levels inhibit the onset of mitosis by interfering with the dephosphorylation of the CDK Tyr15 residue by CDC25.
Taken together, the appearance of multiple phenotypes suggests different roles for the SUC1/CKS1 protein. Amongst these, it may function as a docking factor for both positive and negative regulators of CDK complexes. This model is supported by a recent crystallographic study of a human SUC1/CKS1 homologue, CKSHs1, complexed with CDK2. As a monomer SUC1/CKS1 proteins have a large hydrophobic surface and a cluster of positively charged residues, which represents a putative phosphate anion-binding site. Binding of CKSHs1 to CDK2 involves the hydrophobic surface and positions the anion-binding site close to the substrate recognition site of CDK2, suggesting that CKSHs1 may act in the targeting of CDK2 to already phosphorylated substrates. Both CDC25 and APC are positively regulated by CDK phosphorylation. The observed phenotypes concerning SUC1/CKS1 overexpression and deletion may therefore be a consequence of the inability of the CDK complexes to recognise CDC25 and components of the APC as substrates, with cell cycle arrest as a result.
With respect to cell cycle regulation in plants a summary of the state of the art is given below. In Arabidopsis, thusfar only two CDK genes have been isolated, CDC2aAt and CDC2bAt, of which the gene products share 56% amino acid identity. Both CDKs are distinguished by several features. First, only CDC2aAt is able to complement yeast p34CDC2/CDC28 mutants. Second, CDC2aAt and CDC2bAt bear different cyclin-binding motifs (PSTAIRE and PPTALRE, respectively), suggesting they may bind distinct types of cyclins. Third, although both CDC2aAt and CDC2bAt show the same spatial expression pattern, they exhibit a different cell cycle phase-specific regulation. The CDC2aAt gene is expressed constitutively throughout the whole cell cycle. In contrast, CDC2bAt mRNA levels oscillate, being most abundant during the S and G2 phases.
In addition, multiple cyclins have been isolated from Arabidopsis. The majority displays the strongest sequence similarity with the animal A- or B-type class of cyclins, but also D-type cyclins have been identified. Although the classification of Arabidopsis cyclins is mainly based upon sequence similarity, limited data suggests that this organisation corresponds with differential functions of each cyclin class. Direct binding of any cyclin with an Arabidopsis CDK subunit has, however, not yet been demonstrated.
In order to manage problems related to plant growth, plant architecture and/or plant diseases, it is believed to be of utmost importance to identify and isolate plant genes and gene products involved in the regulation of the plant cell division, and more particularly coding for and interacting with CDK""s and/or their interacting proteins, responsible for the control of the cell cycle and the completion of the S and M phase of the cell cycle. If such novel genes and/or proteins have been isolated and analysed, the growth of the plant as a whole can be influenced. Also, the growth of specific tissues or organs and thus the architecture of the plant can be modified.
In the present invention a two-hybrid screen was exploited to isolate new gene products interacting with CDC2aAt. A positive clone indicative of a hitherto unknown plant cell cycle regulatory nucleotide sequence was identified. A homology search in databases showed the identification of a very first plant homologue of the SUC1 gene from Sch. pombe and the CKS1 gene from S. cerevisiae. Surprisingly the novel plant CKS1 homologue (having less than 50% homology at amino acid level with the corresponding yeast genes) was able to rescue a Sch. pombe temperature-sensitive CDC2 mutant. This confirmed that the newly isolated plant sequence could, also from a functional viewpoint, be designated as a CKS1 homologue. The Arabidopsis gene was designated CKS1At, for CDK-associating subunit from Arabidopsis thaliana. 
Thus a novel plant nucleotide sequence and polypeptide sequence, having a molecular weight of about 10.5 kDa, are provided.
The DNA sequence of CKS1At comprises the nucleotide sequence defined in SEQ.ID NO.1 encoding for a protein as defined in SEQ.ID.NO.3 or for a protein having substantially the same amino acid sequence as the protein defined in SEQ.ID.NO.3.
The coding nucleotide sequence for CKS1At in SEQ.ID.NO. 1 starts at the first ATG codon (position 1) and terminates at codon AAG (position 261).
Using a nucleic acid amplification technology, such as the polymerase chain reaction (PCR), a genomic DNA fragment containing introns was isolated comprising the sequence defined in SEQ.ID.NO. 2.
Thus the invention provides an isolated and/or recombinant nucleic acid molecule, preferably DNA, encoding at least a functional part of a plant CKS1 protein, which protein in Arabidopsis thaliana comprises the sequence as depicted in SEQ.ID.NO.3 or SEQ.ID.NO.4 or a functional part thereof.
A further part of the invention is a nucleic acid molecule comprising at least a part of the sequence as depicted in SEQ.ID.NO.1 or SEQ.ID.NO.2 or a sequence substantially homologous thereto. In a preferred embodiment, this nucleic acid molecule is isolated from a monocotyledonous or dicotyledonous plant species.
A further embodiment of the current invention is a nucleic acid molecule comprising at least a part of the sequence as depicted in SEQ.ID.NO.1 or SEQ.ID.NO.2 or a sequence which hybridizes under conventional, preferably under stringent, conditions to at least a part of said sequence or its complementary sequence.
Alternatively, the nucleotide sequence depicted in SEQ.ID.NO.1 or SEQ.ID.NO.2 can be used to design so-called amplification primers for use in a nucleic acid amplification technique. Said primers can be used in a particular amplification technique to identify and isolate substantially homologous nucleic acid molecules from other plant species. The design and use of said primers is known by a person skilled in the art. Preferably such amplification primers comprise a contiguous sequence of at least 6 nucleotides, in particular 13 nucleotides or more, identical or complementary to the nucleotide sequence depicted in SEQ.ID.NO.1 or SEQ.ID.NO.2.
In addition, the nucleic acid molecule provided in SEQ.ID.NO.1 or SEQ.ID.NO.2 or parts of these sequences can be used to select substantially homologous sequences present in other plants than Arabidopsis thaliana. It has been shown according to the invention that for instance riboprobes from CKS1At hybridize with CKS1 RNA from different plant species.
The Arabidopsis thaliana polypeptide according to the invention comprises the amino acid sequence as defined in SEQ.ID.NO.3.
CKS1At protein binds, in vitro and in vivo, to CDKs such as CDC2aAt and CDC2bAt. The CKS1At protein can also be used to complement Sch.pombe SUC1 disruptants. Furthermore the CKS1At protein can be used to rescue a Sch.pombe temperature-sensitive CDC2 mutant.
Therefore a further part of the invention are polypeptides, preferably plant polypeptides which have, compared to the CKS1At protein, comparable or identical characteristics in terms of binding to cyclin dependent kinases, in particular plant cyclin dependent kinases.
To the scope of the current invention also belong plant polypeptides which have, compared to the CKS1At protein, similar properties to complement Sch.pombe SUC1 disruptants and/or to rescue Sch.pombe temperature-sensitive CDC2 mutants such as the CDC2-L7 strain.
Fragments of the above mentioned polypeptide, such as the first 72 amino acids as illustrated in SEQ.ID.NO. 4, also belong to the invention. The last 15 amino acids of CKS1At, including the polyglutamine stretch, are dispensable for the binding of both CDC2aAt and CDC2bAt. It is likely that these amino acids are involved in interactions with other proteins.
The CKS1At mutant E61Q (which means a mutant protein where at position 61 of the wild type CKS1At, the Glu residue is replaced by the Gln residue) has reduced binding affinity for CDKs. Overexpression of the CKS1At protein caused a G2-specific cell cycle arrest in fission yeast. In contrast, the E61Q mutated protein does not arrest cell cycle progression. This demonstrates that the E61-residue is an important amino acid in CKS1At for interaction with CDKs. A second point mutation (P62G, replacement of proline at position 62 by glycine) in CKS1At also showed reduced binding activity for CDK. The use of this inactive mutants to modulate the cell cycle in plant cells, plant tissues, plant organs or whole plants is part of this invention.
Increased expression levels in maturing leaves indicate a role for CKS1At in endoreduplication, whereas the lack of CDC2aAt and CDC2bAt expression in these tissues suggest the presence of an as yet unidentified CDK protein in Arabidopsis, specifically involved in endoreduplication. These results suggest that CKS1At can also interact with a novel, as yet unidentified CDK protein in Arabidopsis.
Part of the invention is also a polypeptide comprising at least a functional part of a plant CKS1 protein encoded by a nucleic acid sequence comprised in a nucleic acid molecule according to the invention. An example for this is that the polypeptide or a fragment thereof according to the invention is embedded in another amino acid sequence.
To the scope of the present invention also belong numerous variations on the disclosed sequences which could be prepared by those skilled in the art using known techniques. The polypeptides encoded by the nucleic acid sequences above mentioned may be modified by varying their amino acid sequence without substantially altering their function. Derivatives of the polypeptides disclosed herein, such as polypeptides carrying single or multiple amino acid substitutions, deletion and/or additions, are included within the present invention.
A further part of the invention is a polypeptide comprising at least a part of the sequence as provided in SEQ.ID.NO3 or SEQ.ID.NO4 or a polypeptide with at least 40%, and preferably more than 69% homology at amino acid level, such sequence preferably being a plant polypeptide.
Plant cell division can conceptually be influenced in three ways (i) inhibiting or arresting cell division, (ii) maintaining, facilitating or stimulating cell division or (iii) uncoupling DNA synthesis from mitosis and cytokinesis. Being able to uncouple S phase from M phase would create opportunities to inhibit or stimulate the level of endoreduplication in specific cells, tissues and/or organs from living organisms, and more in particular in plant cells, plant tissues, plant organs or whole plants.
To analyse the industrial applicabilities of CKS1At and any plant homologue, for the first time transformed plants overproducing CKS1At were created. Surprisingly, the transformed plants do show modulated endoreduplication. To further analyse whether other plant cell cycle genes could also modulate endoreduplication in plants, transformed plants overproducing a plant cyclin dependent kinase were created, and more in particular plants overexpressing a plant specific dependent kinase such as CDC2b from Arabidopsis thaliana were created. Surprisingly, modulated (and more particular enhanced) endoreduplication could clearly be demonstrated in these transformed plants. In yet an alternative set of experiments, transformed plants expressing a dominant negative mutant of a cyclin dependent kinase were created. More in particular, plants were created which express a mutant cyclin dependent kinase still able to bind to other regulatory cell cycle proteins but with no or limited activity. Even more surprisingly, also these transformed plants demonstrated a significantly modulated level of endoreduplication in comparison with control plants.
Therefore part of this invention is the use of plant cell cycle genes and/or plant cell cycle proteins to modulate endoreduplication in plant cells, plant tissues, plant organs and/or whole plants. The man skilled in the art can use cell cycle genes and proteins from other organisms such as yeast and animals to modulate endoreduplication in plant cells, plant tissues, plant organs and/or whole plants since the functionality of plant cell cycle genes and proteins to modulate endoreduplication is herewith disclosed. The use of these genes and proteins to modulate endoreduplication is therefore also an embodiment of this invention.
In a further preferred embodiment endoreduplication in plant cells, plant tissues, plant organs or whole plants is modulated via enhancing or reducing the expression and/or the activity of a CKS1 gene or CKS1 gene product, preferably a plant CKS1 gene or plant CKS1 gene product. In a further preferred embodiment overexpression of CKS1At is used to enhance endoreduplication in plant cells, plant tissues, plant organs or whole plants.
In yet another preferred embodiment, cyclin dependent kinases, preferably plant cyclin dependent kinase and more preferably plant specific cyclin dependent kinase such as CDC2b from Arabidopsis thaliana, are used to modulate endoreduplication in plant cells, plant tissues, plant organs and/or whole plants. In a further preferred embodiment overexpression of a CDC2b from Arabidopsis is used to enhance endoreduplication in plant cells, plant tissues, plant organs or whole plant.
In a further preferred embodiment expression of a dominant negative mutant of cyclin dependent kinases, such as mutant cyclin dependent kinases which still have binding activities for regulatory cell cycle proteins but have no or only limited kinase activity, is used to modulate endoreduplication in plant cells, plant tissues, plant organs and/or whole plants. One example of such dominant negative mutant is an Arabidopsis thaliana CDC2b variant wherein the aspartic acid at position 161 is replaced by asparagine.
In a further preferred embodiment, expression of the dominant negative mutant of a plant CDC2b is used to modulate the endoreduplication in plant cells, plant tissue, plant organs or whole plants.
Because the present invention for the first time clearly demonstrates that it is possible to modulate endoreduplication in plants or parts thereof by modulating the expression and/or activity of a gene or protein through genetic engineering, the scope of the invention also contemplates a general method for modulating endoreduplication by modifying the expression and/or activity of specific genes or gene products through genetic engineering. A preferred embodiment provides the use of genetic engineering to modulate endoreduplication in plant cells, plant tissue, plant organs and/or whole plants.
With reference to the above, an important aspect of the current invention is a method for modulating endoreduplication in monocotyledonous or dicotyledonous plants or parts thereof by modifying the plant cell division. In a preferred embodiment one or more cell cycle genes or plant cell cycle genes, preferably operably linked to control sequences, are for instance used to specifically modulate endoreduplication in transformed plants, particularly:
in the complete plant
in selected plant organs, tissues or cell types
under specific environmental conditions, including abiotic stress such as cold, heat, drought or salt stress or biotic stress such as pathogen attack
during specific developmental stages.
In a further preferred embodiment, one or more cell cycle genes or plant cell cycle genes, preferably operably linked to a control sequence are used to modulate endoreduplication in storage cells, storage tissues and/or storage organs of plants or parts thereof. Preferred target storage organs and parts thereof for the modulation of endoreduplication according to the invention, are for instance seeds (such as from cereals, oilseed crops), roots (such as in sugar beet), tubers (such as in potato) and fruits (such as in vegetables and fruit species). Furthermore it is expected that increased endoreduplication in storage organs and parts thereof correlates with enhanced storage capacity and as such with improved yield.
In yet another embodiment of the invention, a plant with modulated endoreduplication in the whole plant or parts thereof can be obtained from a single plant cell by transforming the cell, in a manner known to the skilled person, with a cell cycle gene, preferably a plant cell cycle gene and, not necessarily but preferably operably linked to a control sequence. In a preferred embodiment such transformation is performed with a CKS1 gene, a CDC2 gene and/or a dominant negative mutant of a CDC2 gene.
In a further preferred embodiment such transformed plants can be obtained by transforming with a plant CKS1 gene, a plant CDC2 gene and/or a dominant negative mutant of a plant CDC2 gene.
In a further preferred embodiment such transformation is performed with a nucleic acid molecule according to claim 1 and/or the Arabidopsis CDC2b gene and/or a dominant negative mutant of the Arabidopsis CDC2b gene.
Any obtained transformed plant with modulated endoreduplication can be used in a conventional breeding scheme or in in vitro plant propagation to produce more transformed plants with the same characteristics and/or can be used to introduce the same characteristic in other varieties of the same or related species. Such plants are also part of the invention. Seeds obtained from the transformed plants genetically also contain the same characteristic and are part of the invention.
The current invention also demonstrates that CKS1, preferably a plant CKS1 and more preferably CKS1At, can be used to interfere with the plant cell cycle and can be used more specifically to prevent entering mitosis and thus inhibit or even arrest cell division in plants or parts thereof. In a particular preferred embodiment of this invention, cell division is inhibited or arrested in plant meristems.
Alternatively, expression studies (see examples) strongly indicate that in plants or parts thereof, low levels of expression and/or activity of CKS1 are correlated with non-dividing cells. The present invention therefor further embraces a method to use CKS1 preferably a plant CKS1 and more preferably CKS1At, to maintain, facilitate or stimulate cell division in plants or parts thereof. In a particular preferred embodiment, cell division is maintained, facilitated or enhanced in plant meristems. In another preferred embodiment cell division is induced in resting cells.
A further part of this invention is a method for transforming plants with CKS1, preferably plant CKS1 according to the present invention, not necessarily but preferably operably linked to a control sequence. Using this approach, and since cell division is a crucial element in determining the growth and shape of a plant or parts thereof, it is expected that defined modulation of the expression and/or activity of plant CKS1 will allow the production of transformed plants, with modulated growth.
Methods to modify the expression levels and/or the activity of CKS1 preferably plant CKS1, are known to persons skilled in the art and include for instance overexpression, co-suppression, the use of ribozymes, anti-sense strategies, gene silencing approaches.
The invention is in principle applicable to any plant and crop that can be transformed with any of the transformation method known to those skilled in the art and includes for instance corn, wheat, barley, rice, oilseed crops, cotton, tree species, sugar beet, cassava, tomato, potato, numerous other vegetables, fruits.
Similarly, the invention can also be used to modulate the cell division and the growth of cells, preferentially plant cells, in in vitro cultures.
Further in accordance with the invention chimeric genes are provided, comprising the following operably linked polynucleotides:
a. a nucleic acid molecule according to claim 1 to 3
b. one or more control sequences
Alternatively, said chimeric genes comprise the following operably linked polynucleotides:
a. a dominant negative plant CDC2 mutant with characteristics such as the D161N mutant of Arabidopsis CDC2b
b. one or more control sequences
Vectors or expression vectors comprising a nucleic acid molecule according to claim 1 or comprising chimeric genes such as described above are also considered as part of the invention.
Part of the invention is also a plant cell carrying at least a functional part of the nucleic acid molecule according to the invention or a chimeric gene as described above.
The present invention is also directed to a transgenic plant carrying a plant cell comprising a nucleic acid molecule according to claim 1 or a chimeric gene as described above.
A transgenic plant is obtained through a process of regenerating said plant starting from a plant cell having as part of its genetic material the nucleic acid molecule according to the invention or a chimeric gene as described above. Progeny of the plant and/or plant material such as flowers, fruit, leaves, pollen, seeds, seedlings or tubes obtainable from said transgenic plant also belong to the current invention.
Also part of the invention are antibodies recognising a plant CKS1 protein or a part thereof. Another part of the invention is the use of antibodies raised against CKS1At to identify and isolate other plant CKS1 proteins and genes.
In order to clarify what is meant in this description by some terms a further explanation is hereunder given.
The polypeptides of the present invention are not necessarily translated from a designated nucleic acid sequence; the polypeptides may be generated in any manner, including for example, chemical synthesis, or expression of a recombinant expression system, or isolation from a suitable viral system. The polypeptides may include one or more analogs of amino acids, phosphorylated amino acids or unnatural amino acids. Methods of inserting analogs of amino acids into a sequence are known in the art. The polypeptides may also include one or more labels, which are known to those skilled in the art.
The terms xe2x80x9cgene(s)xe2x80x9d, xe2x80x9cpolynucleotidexe2x80x9d, xe2x80x9cnucleic acid sequencexe2x80x9d, xe2x80x9cnucleotide sequencexe2x80x9d, xe2x80x9cDNA sequencexe2x80x9d or xe2x80x9cnucleic acid molecule(s)xe2x80x9d as used herein refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule. Thus, this term includes double- and single-stranded DNA, and RNA. It also includes known types of modifications, for example, methylation, xe2x80x9ccapsxe2x80x9d substitution of one or more of the naturally occuring nucleotides with an analog.
xe2x80x9cRecombinant nucleic acid moleculexe2x80x9d as used herein refers to a polynucleotide of genomic, cDNA, semisynthetic or synthetic origin which, by virtue of its origin or manipulation (1) is linked to a polynucleotide other than that to which it is linked in nature or, (2) does not occur in nature.
An xe2x80x9cexpression vectorxe2x80x9d is a construct that can be used to transform a selected host cell and provides for expression of a coding sequence in the selected host. Expression vectors can for instance be cloning vectors, binary vectors or integrating vectors.
A xe2x80x9ccoding sequencexe2x80x9d is a nucleotide sequence which is transcribed into mRNA and/or translated into a polypeptide when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a translation start codon at the 5xe2x80x2-terminus and a translation stop codon at the 3xe2x80x2-terminus. A coding sequence can include, but is not limited to mRNA, cDNA, recombinant nucleotide sequences or genomic DNA, while introns may be present as well under certain circumstances.
xe2x80x9cControl sequencexe2x80x9d refers to regulatory DNA sequences which are necessary to effect the expression of coding sequences to which they are ligated. The nature of such control sequences differs depending upon the host organism. In prokaryotes, control sequences generally include promoter, ribosomal binding site, and terminators. In eukaryotes generally control sequences include promoters, terminators and, in some instances, enhancers, transactivators or transcription factors. The term xe2x80x9ccontrol sequencexe2x80x9d is intended to include, at a minimum, all components the presence of which are necessary for expression, and may also include additional advantageous components.
xe2x80x9cOperably linkedxe2x80x9d refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. A control sequence xe2x80x9coperably linkedxe2x80x9d to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences. In case the control sequence is a promoter, it is obvious for a skilled person that double-stranded nucleic acid is used.
The terms xe2x80x9cproteinxe2x80x9d and xe2x80x9cpolypeptidexe2x80x9d used in this application are interchangeable. xe2x80x9cPolypeptidexe2x80x9d refers to a polymer of amino acids (amino acid sequence) and does not refer to a specific length of the molecule. Thus peptides and oligopeptides are included within the definition of polypeptide. This term does also refer to or include post-translational modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like. Included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), polypeptides with substituted linkages, as well as other modifications known in the art, both naturally occurring and non-naturally occurring.
xe2x80x9cFragment of a sequencexe2x80x9d or xe2x80x9cpart of a sequencexe2x80x9d means a truncated sequence of the original sequence referred to. The truncated sequence (nucleic acid or protein sequence) can vary widely in length; the minimum size being a sequence of sufficient size to provide a sequence with at least a comparable function and/or activity of the original sequence referred to, while the maximum size is not critical. In some applications, the maximum size usually is not substantially greater than that required to provide the desired activity and/or function(s) of the original sequence. Typically, the truncated amino acid sequence will range from about 5 to about 60 amino acids in length. More typically, however, the sequence will be a maximum of about 50 amino acids in length, preferably a maximum of about 30 amino acids. It is usually desirable to select sequences of at least about 10, 12 or 15 amino acids, up to a maximum of about 20 or 25 amino acids.
The term xe2x80x9cantibodyxe2x80x9d includes, without limitation, chimeric antibodies, altered antibodies, univalent antibodies, bi-specific antibodies, Fab proteins or single-domain antibodies. In many cases, the binding phenomena of antibodies to antigens is equivalent to other ligand/anti-ligand binding. The antibody can be a monoclonal or a polyclonal antibody.
xe2x80x9cTransformationxe2x80x9d as used herein, refers to the transfer of an exogenous polynucleotide into a host cell, irrespective of the method used for the transfer. The polynucleotide may be transiently or stably introduced into the host cell and may be maintained non-integrated , for example, as a plasmid, or alternatively, may be integrated into the host genome. Many types of vectors can be used to transform a plant cell and many methods to transform plants are available. Examples are direct gene transfer, pollen-mediated transformation, plant RNA virus-mediated transformation, Agrobacterium-mediated transformation, liposome-mediated transformation, transformation using wounded or enzyme-degraded immature embryos, or wounded or enzyme-degraded embryogenic callus. All these methods and several more are known to persons skilled in the art. The resulting transformed plant cell can then be used to regenerate a transformed plant in a manner known by a skilled person.
xe2x80x9cFunctional part ofxe2x80x9d means that said part to which subject it relates has substantially the same activity as the subject itself, although the form, length or structure may vary.
The term xe2x80x9csubstantially homologousxe2x80x9d refers to a subject, for instance a nucleic acid, which is at least 50% identical in sequence to the reference when the entire ORF (open reading frame) is compared, where the sequence identity is preferably at least 70%, more preferably at least 80%, still more preferably at least 85%, especially more than about 90%, most preferably 95% or greater, particularly 98% or greater. Thus, for example, a new nucleic acid isolate which is 80% identical to the reference is considered to be substantially homologous to the reference. Sequences that are substantially homologous can be identified by comparing the sequences using standard software available in sequence data banks, or in a Southern hybridisation experiment under, for instance, conventional or preferably stringent conditions as defined for that particular system.
Similarly, in a particular embodiment, two amino acid sequences, when proper aligned in a manner known to a skilled person, are xe2x80x9csubstantially homologousxe2x80x9d when more than 40% of the amino acids are identical or similar, or when more preferably more than about 60% and most preferably more than 69% of the amino acids are identical or similar (functionally identical).
xe2x80x9cSense strandxe2x80x9d refers to the strand of a double-stranded DNA molecule that is homologous to a mRNA transcript thereof. The xe2x80x9canti-sense strandxe2x80x9d contains an inverted sequence which is complementary to that of the xe2x80x9csense strandxe2x80x9d.
xe2x80x9cCell cyclexe2x80x9d or xe2x80x9ccell divisionxe2x80x9d means the cyclic biochemical and structural events associated with growth and with division of cells, and in particular with the regulation of the replication of DNA and mitosis. The cycle is divided into periods called: G0, Gap1 (G1), DNA synthesis (S), Gap2 (G2), and mitosis (M).
xe2x80x9cCell cycle genesxe2x80x9d are genes encoding proteins involved in the regulation of the cell cycle or fragments thereof.
xe2x80x9cPlant cell cycle genesxe2x80x9d are cell cycle genes originally present or isolated from a plant or fragments thereof.
xe2x80x9cPlant cellxe2x80x9d comprises any cell derived from any plant and existing in culture as a single cell, a group of cells or a callus. A plant cell may also be any cell in a developing or mature plant in culture or growing in nature.
xe2x80x9cPlantsxe2x80x9d comprises all plants, including monocotyledonous and dicotyledonous plants.
xe2x80x9cPlant sequencexe2x80x9d is a sequence naturally occurring in a plant.
xe2x80x9cPlant polypeptidexe2x80x9d is a polypeptide naturally occurring in a plant.
xe2x80x9cCyclin-dependent protein kinase complexxe2x80x9d means the complex formed when preferably functional, cyclin associates with a, preferably, functional cyclin dependent kinase. Such complexes may be active in phosphorylating proteins and may or may not contain additional protein species.
xe2x80x9cCell-cycle kinase inhibitorxe2x80x9d (CKI) is a protein which inhibit CDK/cyclin activity and is produced and/or activated when further cell division has to be temporarily or continuously prevented.
xe2x80x9cExpressionxe2x80x9d means the production of a protein or nucleotide sequence in the cell itself or in a cell-free system. It includes transcription into an RNA product, post-transcriptional modification and/or translation to a protein product or polypeptide from a DNA encoding that product, as well as possible post-translational modifications.
xe2x80x9cModulation of expression or activityxe2x80x9d means control or regulation, positively or negatively, of the expression or activity of a particular protein or nucleotide sequence by methods known to a skilled person.
xe2x80x9cEndoreduplicationxe2x80x9d means recurrent DNA replication without consequent mitosis and cytokinesis.
xe2x80x9cForeignxe2x80x9d with regard to a DNA sequence means that such a DNA is not in the same genomic environment in a cell, transformed with such a DNA in accordance with this invention, as is such DNA when it is naturally found in a cell of the plant, bacteria, fungus, virus or the like, from which such a DNA originates.
In the description of the current invention reference is made to the following sequences of the Sequence Listing:
SEQ.ID.NO. 1: coding nucleotide sequence (position 1-261) for CKS1At with flanking non-coding sequences.
SEQ.ID.NO. 2: genomic nucleotide sequence with introns
SEQ.ID.NO. 3: amino acid sequence (position 1-87) obtainable from the coding nucleotide sequence represented in SEQ.ID.NO. 1.
SEQ.ID.NO.4: amino acid sequence (position 1-72) obtainable from the coding nucleotide sequence represented in SEQ.ID.NO. 1.